Protein denatured states remain poorly understood and yet, as the starting point for protein folding, an understanding of the conformational constraints acting upon the denatured state is central to solving the problem of how a protein folds efficiently. Misfolding diseases,such as Alzheimer's and Parkinson's diseases are major health problems, where the causative agents are believed to be non-native and denatured states of proteins. Thus, fundamental research on denatured proteins is essential to new insight into the genesis of these disease states. This laboratory has developed a novel strategy to probe the conformational and thermodynamic properties of unfolded proteins. The propensity for forming loops of different sizes is assessed through histidine-heme loop equilibria. Single surface histidine variants have been produced in yeast iso-1-cytochrome c, allowing loop equilibria for loops of 9 to 83 amino acids to be measured under denaturing conditions. Formation of closed loops are required in the earliest stages of structure accretion when a protein folds. This system has already yielded important insights into the deviation of protein denatured states from random coil behavior. Several key properties of unfolded proteins will be probed with this system. To understand how residual structure affects contact probability in a denatured protein, we will stabilize residual structure with disulfide crosslinks, insert a known stable beta hairpin into iso-1-cytochrome c, and apply our methodology to cytochrome c', which is know to have a much more compact denatured state than iso-1-cytochrome c (specific aim 1). The effect of sequence composition on denatured state conformational properties will be probed by inserting homopolymeric arnino acid sequences into the disordered N-terminal region of iso-1-cytochrome c (specific aim 2) with emphasis on the properties of the flexible amino acid glycine and the rigid amino acid proline. Kinetics studies on loop formation and breakage are planned to probe how loop size, denatured state compactness and residual structureimpact the rate at which denatured state contacts form and the factors which cause contacts to persist (specific aim 3). NMR and FRET methods will be used to correlate denatured state thermodynamic with denatured state structural properties (specific aim 4). Our multipronged approach probes key parameters of protein denatured states expectedto be principal modulators of early events in protein folding.